Abstract/Project Summary. Opioid abuse and addiction are major public health concerns. Opioids are highly useful analgesics, yet an estimated two million people in the United States suffer disorders related to abuse of prescription opioids. The social and economic costs of these disorders are devastating and on the rise: an average of 44 people die every day from prescription opioid overdoses. Opioids are structurally diverse molecules that include oxycodone, heroin and endorphins, and the physiological effects of these molecules are mediated by G protein-coupled receptors (GPCRs). Recently developed `biased' opioid agonists demonstrate that this diversity can be mined to identify drugs with less harmful side effects, but the mechanism of action of these `biased' agonists remains incompletely resolved. New technical advances now make it possible to biochemically capture the protein interaction networks mediating GPCR activity from inside of living cells with sub-minute temporal resolution. The ability to capture, quantify, and characterize the endogenous proteins which mediate and regulate opioid activity opens new doors for determining how biased agonists differ from endogenous ligands or drugs of abuse. This K99/R00 award combines critical new training in cutting-edge proteomics with traditional cell biological techniques to examine the mechanism of biased opioid agonism: Aim 1-Define the kinetics by which different classes of opioids alter mu opioid receptor (OR) location and coupling to its transducer and regulatory proteins; Aim 2-Identify new protein regulators of OR stimulated by standard or biased opioids. Aim 3- Define OR signaling targets and determine if these proteins differ between opioids. Future studies based on these results will help to define how opioid receptors operate under normal, pharmacologically activated, or disease states resulting from drug abuse and addiction.